Epinephrine nanoparticles, methods of fabrication thereof, and methods for use thereof for treatment of conditions responsive to epinephrine

ABSTRACT

The invention provides compositions including epinephrine nanoparticles and methods for therapeutic use of the compositions in the treatment of conditions responsive to epinephrine such as a cardiac event or an allergic reaction, particularly anaphylaxis. The epinephrine nanoparticles can be incorporated into orally-disintegrating and fast-disintegrating tablet pharmaceutical formulations and can significantly increase the sublingual bioavailability of epinephrine, and thereby reduce the epinephrine dose required. Additionally, the invention provides methods for fabrication of stabilized epinephrine nanoparticles for use in the described compositions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application No. 61/550,359, filed on Oct. 21, 2011, the contentof which is hereby incorporated by reference in its entirety.

This application is related to U.S. Provisional Patent Application No.61/660,273, filed on Jun. 15, 2012.

This application is related to International Application No.PCT/US2011/26604, filed on Mar. 1, 2011, which claims the benefit ofU.S. Provisional Patent Application No. 61/309,136, filed on Mar. 1,2010.

This application is related to U.S. Provisional Patent Application No.60/715,180, filed on Sep. 9, 2005, and U.S. Provisional PatentApplication No. 60/759,039, filed on Jan. 17, 2006.

This application is also related to U.S. Utility patent application Ser.No. 11/672,503, filed on Feb. 7, 2007, now abandoned, which is acontinuation-in-part of U.S. Utility patent application Ser. No.11/530,360, filed on Sep. 8, 2006, now abandoned.

The aforementioned provisional and utility applications are herebyincorporated by reference in their entireties. The informationincorporated is as much a part of the instant application as filed as ifthe text was repeated in the application, and should be treated (theincorporated information) as part of the text of the application asfiled.

FIELD OF THE INVENTION

The invention generally relates to compositions and methods fortreatment of conditions responsive to epinephrine (also known asadrenaline), particularly to compositions and methods for emergencytreatment of conditions responsive to epinephrine, and most particularlyto compositions including epinephrine nanoparticles for sublingualadministration in treatment of conditions responsive to epinephrine.

BACKGROUND

Tablets that disintegrate or dissolve rapidly in the patient's mouthwithout the use of water are convenient for the elderly, young children,patients with swallowing difficulties, and in situations where water isnot available. For these specially designed formulations, the smallvolume of saliva that is available is sufficient to disintegrate ordissolve a tablet in the oral cavity. The drug released from thesetablets can be absorbed partially or entirely into the systemiccirculation from the buccal mucosa or sublingual cavity, or can beswallowed as a solution to be absorbed from the gastrointestinal tract.

The sublingual route usually produces a faster onset of action thantraditional orally administered tablets and the portion absorbed throughthe sublingual blood vessels bypasses the hepatic first pass metabolicprocesses (Birudaraj et al., 2004, J Pharm Sci 94; Motwani et al., 1991,Clin Pharmacokinet 21: 83-94; Ishikawa et al., 2001, Chem Pharm Bull 49:230-232; Price et al., 1997, Obstet Gynecol 89: 340-345; Kroboth et al.,1995, J Clin Psychopharmacol 15: 259-262; Cunningham et al., 1994, JClin Anesth 6: 430-433; Scavone et al., 1992, Eur J Clin Pharmacol 42:439-443; Spenard et al., 1988, Biopharm Drug Dispos 9: 457-464).

Likewise, due to high buccal and sublingual vascularity, buccally- orsublingually-delivered drugs can gain direct access to the systemiccirculation and are not subject to first-pass hepatic metabolism. Inaddition, therapeutic agents administered via the buccal or sublingualroute are not exposed to the acidic environment of the gastrointestinaltract (Mitra et al., 2002, Encyclopedia of Pharm. Tech., 2081-2095).Further, the buccal and sublingual mucosas have low enzymatic activityrelative to the nasal and rectal routes. Thus, the potential for druginactivation due to biochemical degradation is less rapid and extensivethan other administration routes (de Varies et al., 1991, Crit. Rev.Ther. Drug Carr. Syst. 8: 271-303).

The buccal and sublingual mucosas are also highly accessible, whichallows for the use of tablets which are painless, easily administered,easily removed, and easily targeted. Because the oral cavity consists ofa pair of buccal mucosa, tablets, such as fast disintegrating tablets,can be applied at various sites either on the same mucosa or,alternatively, on the left or right buccal mucosa (Mitra et al., 2002,Encyclopedia of Pharm. Tech., 2081-2095). In addition, the buccal andsublingual routes could be useful for drug administration to unconsciouspatients, patients undergoing an anaphylactic attack, or patients whosense the onset of an anaphylactic attack.

Anaphylaxis is a sudden, severe systemic allergic reaction, which can befatal within minutes. Epinephrine (Epi) is the drug of choice for thetreatment of anaphylaxis worldwide (Joint Task Force on PracticeParameters, 2005, J Allergy Clin Immunol 115: S483-S523; Lieberman,2003, Curr Opin Allergy Clin Immunol 3: 313-318; Simons, 2004, J AllergyClin Immunol 113: 837-844). It is available as an injectable dosage formin ampoules or in autoinjectors, however these are underused whenanaphylaxis occurs (Simons, F. E. R. J Allergy Clin Immunol124(4):625-636 2009; Simons, F. E. R. J Allergy Clin Immunol125:S161-181 2010). The drawbacks of Epi autoinjectors include highcost, perceived large size and bulkiness, limitations on repeated dosing(if required), fear and anxiety associated with the use of needles(especially in children), and dosing errors caused by incorrecttechniques of administration (Simons, K. J. et al. Current Opinion inClinical Immunology 10:354-361 2010). Furthermore, in aqueous solutions,epinephrine is unstable in the presence of light, oxygen, heat, andneutral or alkaline pH values (Connors et al., 1986, in ChemicalStability of Pharmaceuticals: A Handbook for Pharmacists,Wiley-Interscience Publication: New York).

The sublingual route of administration is a promising alternative routefor epinephrine administration. The formulation of sublingual tablets ofepinephrine would enable the development of tablets with a range ofepinephrine doses to match the population on a mg/kg basis. Sublingualtablets of epinephrine would be easy to carry and self-administereliminating the fear and anxiety associated with needles used inautoinjectors for young children, as well as readily providing thecapability of multiple doses. Feasibility studies in humans and animalshave shown that epinephrine can be absorbed sublingually (Gu et al.,2002, Biopharm Drug Dispos 23: 213-216; Simons et al., 2004, J AllergyClin Immunol 113: 425-438). The recommended dose of epinephrine for thetreatment of anaphylaxis is about 0.01 mg/Kg: usually about 0.2 mL toabout 0.5 mL of a 1:1000 dilution of epinephrine in a suitable carrier.Based on historical and anecdotal evidence, an approximately 0.3 mg doseof epinephrine, by subcutaneous (SC) or intramuscular (IM) injectioninto the deltoid muscle, has been agreed upon as the dose required forthe emergency treatment of anaphylaxis. Recent studies have demonstratedthat if the approximately 0.3 mg dose is administered IM into thelaterus vascularis (thigh) muscle, Epi plasma concentrations are higherand occur more quickly than SC or IM administration into the deltoidmuscle. (Joint Task Force on Practice Parameters, 2005, J Allergy ClinImmunol 115: S483-S523; Lieberman, 2003, Curr Opin Allergy Clin Immunol3: 313-318; Simons, 2004, J Allergy Clin Immunol 113: 837-844)).

As stated above, epinephrine (Epi) is typically administered eithersubcutaneously (SC) or intramuscularly (IM) by injection. Thus, Epiinjections are the accepted first aid means of delivering Epi and areadministered either manually or by automatic injectors. It isrecommended that persons at risk of anaphylaxis, and persons responsiblefor children at risk for anaphylaxis, maintain one or more automatic Epiinjectors in a convenient place at all times.

Given the difficulties associated with manual subcutaneous (SC) orintramuscular (IM) administration of Epi, such as patient apprehensionrelated to injections or the burden of an at risk person having toalways maintain an Epi injector close at hand, there exists a need inthe art for more convenient dosage forms which can provide immediateadministration of Epi, particularly to a person undergoing anaphylaxiswherein the need for injection or Epi injectors is obviated.

Recently, a novel fast-disintegrating tablet suitable for sublingual(SL) administration was developed. See related U.S. applications: U.S.Provisional Patent Application No. 60/715,180; U.S. Provisional PatentApplication No. 60/759,039; U.S. Utility patent application Ser. No.11/672,503; and U.S. Utility patent application Ser. No. 11/530,360.Sublingual administration of 40 mg epinephrine as the bitartrate saltusing these novel tablets resulted in a rate and an extent ofepinephrine absorption similar to that achieved following intramuscularinjections of 0.3 mg epinephrine in the thigh. Sublingual doses rangingfrom 5 to 40 mg epinephrine as the bitartrate salt were studied toachieve equivalent plasma concentrations. In an animal model, it wasdetermined that a 40 mg epinephrine dose administered sublingually as abitartrate salt in tablet form resulted in plasma epinephrineconcentrations similar to those achieved by 0.3 mg epinephrineintramuscular (IM) injection (Rawas-Qalaji et al. J Allergy Clin Immunol117:398-403 2006).

Without being bound by theory, it is thought that fabrication ofepinephrine into nanoparticles and incorporation of the nanoparticlesinto a tablet formulation with pharmaceutically-acceptable carriers,penetration enhancers, and mucoadhesives will significantly increase theabsorption of SL-administered epinephrine and will result in thereduction of SL epinephrine dose required.

SUMMARY OF THE INVENTION

Epinephrine (Epi) is life-saving in the treatment of anaphylaxis. Incommunity settings, a first-aid dose of epinephrine in an amount of 0.15mg or 0.3 mg is injected into the mid-outer thigh by patients orcaregivers using an auto-injector such as an EpiPen® (epinephrineauto-injector 0.3/0.15 mg, Dey Pharma, L.P. Nappa, Calif.). Epiauto-injectors are under-used because of needle phobia, bulky size, andhigh cost; additionally, there are only two fixed doses, shelf-life isonly 12-18 months, and unintentional injection and injury sometimesoccur.

The instant invention circumvents the aforementioned problems byproviding a fast-disintegrating epinephrine tablet formulation foranaphylaxis treatment. Although this formulation was designed withregard to anaphylaxis, it is equally effective and contemplated for usein treatment of any condition responsive to epinephrine such as cardiacevents, i.e. cardiac arrest, and breathing difficulties, i.e. asthma,bronchial asthma, bronchitis, emphysema, and respiratory infections.

In a validated rabbit model, this fast-disintegrating epinephrine tabletformulation resulted in plasma epinephrine concentrations similar tothose achieved after a 0.3 mg epinephrine intra-muscular injection(Rawas-Qalaji et al. J Allergy Clin Immunol 117:398-403 2006).Furthermore, epinephrine was stable in these fast-disintegrating tabletsfor at least seven years.

In one aspect, the invention provides epinephrine nanoparticles. Theepinephrine can be either an epinephrine base or an epinephrinebitartrate salt.

The invention also provides stabilized epinephrine nanoparticles.

In another aspect, the invention provides a composition, includingepinephrine nanoparticles, capable of enhancing the sublingualbioavailability of epinephrine for the emergency treatment ofanaphylaxis.

The invention additionally provides a method for fabrication ofstabilized epinephrine nanoparticles and incorporation of the fabricatednanoparticles into orally-disintegrating and fast-disintegratingtablets. The fabrication method includes combining a pre-determinedamount of epinephrine (epinephrine base or epinephrine bitartrate salt)and a solvent in a reaction chamber to form a mixture and exposing themixture to at least one pass at a pre-determined pressure and apre-determined temperature. The pre-determined pressure ranges fromabout 8,000 psi to 30,000 psi. The pre-determined temperature rangesfrom 8.3 to 43.3° C. The solvent with which the epinephrine is combinedcan be water with or without sodium metabisulfite, isopropyl alcohol(ISP), methanol, acetonitrile, acetone, hexane, chloroform,dichloromethane (DCM), tetrahydrofuran (THF), ethyl acetate, phosphoricacid, and acetic acid.

As described herein, buccal or sublingual oral disintegrating tablets(ODTs) are distinguished from conventional sublingual tablets, lozenges,or buccal tablets by the ODTs' ability to fully dissolve or disintegratein less than about one minute in the mouth.

The fabrication method for stabilized epinephrine nanoparticles can alsoinclude exposing the mixture of epinephrine and solvent to a second passat a different pre-determined pressure and a different pre-determinedtemperature from that of the first pass.

Additionally, nanoparticles fabricated by the method can be lyophilized(freeze-dried) or dried under vacuum.

In another aspect, the invention provides a pharmaceutical composition,including epinephrine nanoparticles, formulated for buccal or sublingualadministration.

The invention also provides a pharmaceutical composition, includingepinephrine nanoparticles, and a pharmaceutically-acceptable carrier forbuccal or sublingual administration.

The phrase “pharmaceutically-acceptable carrier” refers to an inactiveand non-toxic substance used in association with an active substance,i.e. epinephrine, especially for aiding in the application of the activesubstance. Non-limiting examples of pharmaceutically-acceptable carriersare diluents, binders, disintegrants, flavorings, fillers, andlubricants. Pharmaceutically-acceptable carriers can have more than onefunction, i.e. a filler can also be a disintegrant. Additionally,pharmaceutically-acceptable carriers may also be referred to asnon-medicinal ingredients (NMIs).

The invention also provides a pharmaceutical composition, for buccal orsublingual administration, including epinephrine nanoparticles and atleast one of a pharmaceutically-acceptable carrier, a penetrationenhancer, and a mucoadhesive. The pharmaceutical composition can furtherinclude at least one of a taste enhancer and a sweetening agent andmouthfeel enhancer. A non-limiting example of a taste enhancer is citricacid. Citric acid masks the bitter taste of epinephrine. A non-limitingexample of a sweetening agent and mouthfeel enhancer is mannitol. Thepharmaceutical composition can further include at least one of a filler,a lubricant, and a disintegrant. Non-limiting examples includemicrocrystalline cellulose (filler), magnesium stearate (lubricant), andhydroxypropyl ethers of cellulose (disintegrant).

Additionally, the invention provides a pharmaceutical compositionincluding epinephrine nanoparticles, in which the bitter taste of theepinephrine is masked by a taste enhancer. A non-limiting example of ataste enhancer is citric acid.

In another aspect, the invention provides a method for enhancingsublingual bioavailability of epinephrine in a subject in need thereofincluding steps for providing a composition including epinephrinenanoparticles and at least one pharmaceutically-acceptable carrier andadministering the composition to the subject. The describedfast-disintegrating epinephrine tablets enhance bioavailability ofepinephrine by releasing epinephrine within sixty seconds ofadministration.

In another aspect, the invention provides a method for treating acondition responsive to epinephrine in a subject in need thereofincluding steps for providing a composition including epinephrinenanoparticles and at least one pharmaceutically-acceptable carrier andadministering the composition to the subject. Conditions responsive toepinephrine react to administration of epinephrine. Non-limitingexamples of conditions responsive to epinephrine include a cardiacevent, i.e. cardiac arrest, or an allergic reaction, i.e. anaphylaxis,asthma, or bronchial asthma.

The phrase “effective amount” refers to the amount of a compositionnecessary to achieve the composition's intended function.

The phase “therapeutically-effective amount” refers to the amount of acomposition required to achieve the desired function, i.e. treatment ofthe condition responsive to epinephrine.

In another aspect, the invention provides a method for treating abreathing difficulty in a subject in need thereof including steps forproviding a composition including epinephrine nanoparticles and at leastone pharmaceutically-acceptable carrier and administering thecomposition to the subject. Breathing difficulties responsive toepinephrine include, but are not limited to, breathing difficultiesassociated with anaphylaxis, asthma, bronchial asthma, bronchitis,emphysema, and respiratory infections.

The invention additionally provides a method for treatment of anallergic emergency in a subject diagnosed with or suspected of having anallergic emergency including steps for providing a composition includingepinephrine nanoparticles and at least one pharmaceutically-acceptablecarrier and administering the composition to the subject. Non-limitingexamples of allergic emergencies are anaphylaxis, asthma, and bronchialasthma.

In an additional aspect, the invention provides a method for treatmentof a cardiac event in a subject diagnosed with or suspected of having acardiac event including steps for providing a composition includingepinephrine nanoparticles and at least one pharmaceutically-acceptablecarrier and administering the composition to the subject. A non-limitingexample of a cardiac event is cardiac arrest.

In another embodiment, the invention provides epinephrine nanoparticlesincluding chitosan and tripolyphosphate (TPP). The epinephrine can be anepinephrine bitartrate salt.

The invention additionally provides epinephrine nanoparticlesencapsulated with chitosan and tripolyphosphate (TPP).

The invention additionally provides a pharmaceutical compositionincluding epinephrine nanoparticles encapsulated with chitosan andtripolyphosphate (TPP).

The invention additionally provides epinephrine nanoparticles includingor encapsulated with chitosan, tripolyphosphate (TPP), and a tasteenhancer. In one embodiment, the taste enhancer is citric acid. Thecitric acid masks the bitter taste of epinephrine. The inventionadditionally encompasses a pharmaceutical composition comprisingepinephrine nanoparticles including or encapsulated with chitosan,tripolyphosphate (TPP), and a taste enhancer.

The invention additionally provides epinephrine nanoparticles includingor encapsulated with chitosan, tripolyphosphate (TPP), and a sweeteningagent and mouthfeel enhancer. In one embodiment, the sweetening agentand mouthfeel enhancer is mannitol. The invention additionallyencompasses a pharmaceutical composition comprising epinephrinenanoparticles including or encapsulated with chitosan, tripolyphosphate(TPP), and a sweetening agent and mouthfeel enhancer.

The invention additionally provides epinephrine nanoparticles includingor encapsulated with chitosan, tripolyphosphate (TPP), and at least oneof a taste enhancer, and a sweetening agent and mouthfeel enhancer. Inone embodiment, the taste enhancer is citric acid and the sweeteningagent and mouthfeel enhancer is mannitol. The invention additionallyencompasses a pharmaceutical composition comprising epinephrinenanoparticles including or encapsulated with chitosan, tripolyphosphate(TPP), and at least one of a taste enhancer, and a sweetening agent andmouthfeel enhancer.

The invention provides a pharmaceutical composition, comprisingepinephrine nanoparticles including or encapsulated with chitosan andtripolyphosphate (TPP), capable of enhancing the sublingualbioavailability of epinephrine, particularly in the emergency treatmentof anaphylaxis.

Any of the above-disclosed epinephrine nanoparticles, compositions, andpharmaceutical compositions can be formulated for buccal or sublingualadministration, particularly those epinephrine nanoparticles,compositions, and pharmaceutical compositions intended for use inemergency treatments.

The invention also provides a method for fabrication of stabilizedepinephrine nanoparticles including or encapsulated with chitosan andtripolyphosphate (TPP) and incorporation of the fabricated nanoparticlesinto orally-disintegrating and fast-disintegrating tablets. The methodfor fabrication includes preparing a first solution including chitosan,acetic acid, and water; preparing a second solution includingtripolyphosphate (TPP) and epinephrine; and adding the second solutionto the first solution and mixing the solutions together. The method forfabrication can additionally include steps for adding at least one of ataste enhancer and a sweetening agent and mouthfeel enhancer to thesolutions. In one embodiment, the taste enhancer is citric acid and thesweetening agent and mouthfeel enhancer is mannitol.

Also encompassed within the invention are epinephrine nanoparticlesproduced by the fabrication method and compositions includingepinephrine nanoparticles produced by the fabrication method.

The invention also provides a pharmaceutical composition includingepinephrine nanoparticles, chitosan, tripolyphosphate (TPP), and atleast one of a pharmaceutically-acceptable carrier, penetrationenhancers, and mucoadhesives for buccal or sublingual administration.

The invention also provides a pharmaceutical composition comprisingepinephrine nanoparticles including chitosan, tripolyphosphate (TPP),and at least one of a taste enhancer and a sweetening agent andmouthfeel enhancer. In one embodiment, the taste enhancer is citric acidand the sweetening agent and mouthfeel enhancer is mannitol.

The invention also provides a pharmaceutical composition includingepinephrine nanoparticles, chitosan, tripolyphosphate (TPP), and atleast one of a pharmaceutically-acceptable carrier, penetrationenhancers, mucoadhesives, taste enhancers, and a sweetening agent andmouthfeel enhancer for buccal or sublingual administration.

The invention also provides a pharmaceutical composition includingepinephrine nanoparticles, in which the bitter taste of epinephrine ismasked by a taste enhancer. In one embodiment, the taste enhancer iscitric acid.

In another aspect, the invention provides a method for enhancingsublingual bioavailability of epinephrine in a subject in need thereofincluding steps for providing a composition including epinephrinenanoparticles encapsulated with chitosan and tripolyphosphate (TPP) andat least one pharmaceutically-acceptable carrier and administering thecomposition to the subject. The described fast-disintegratingepinephrine tablets enhance bioavailability of epinephrine by releasingepinephrine within sixty seconds of administration.

In another aspect, the invention provides a method for treating acondition responsive to epinephrine in a subject in need thereofincluding steps for providing a composition including epinephrinenanoparticles encapsulated with chitosan and tripolyphosphate (TPP) andat least one pharmaceutically-acceptable carrier and administering thecomposition to the subject. Conditions responsive to epinephrine reactto administration of epinephrine. Non-limiting examples of conditionsresponsive to epinephrine include a cardiac event, i.e. cardiac arrest,or an allergic reaction, i.e. anaphylaxis, asthma, or bronchial asthma.

In another aspect, the invention provides a method for treating abreathing difficulty in a subject in need thereof including steps forproviding a composition including epinephrine nanoparticles encapsulatedwith chitosan and tripolyphosphate (TPP) and at least onepharmaceutically-acceptable carrier and administering the composition tothe subject. Breathing difficulties responsive to epinephrine include,but are not limited to, breathing difficulties associated withanaphylaxis, asthma, bronchial asthma, bronchitis, emphysema, andrespiratory infections.

The invention additionally provides a method for treatment of anallergic emergency in a subject diagnosed with or suspected of having anallergic emergency including steps for providing a composition includingepinephrine nanoparticles encapsulated with chitosan andtripolyphosphate (TPP) and at least one pharmaceutically-acceptablecarrier and administering the composition to the subject. Non-limitingexamples of allergic emergencies are anaphylaxis, asthma, and bronchialasthma.

In an additional aspect, the invention provides a method for treatmentof a cardiac event in a subject diagnosed with or suspected of having acardiac event including steps for providing a composition includingepinephrine nanoparticles encapsulated with chitosan andtripolyphosphate (TPP) and at least one pharmaceutically-acceptablecarrier and administering the composition to the subject. A non-limitingexample of a cardiac event is cardiac arrest.

In another aspect, any of the above-disclosed epinephrine nanoparticlescan be used in the manufacture of any of the above-disclosedcompositions and pharmaceutical compositions.

Other objectives and advantages of this invention will become apparentfrom the following description taken in conjunction with theaccompanying drawings, wherein are set forth, by way of illustration andexample, certain embodiments of this invention. The drawings constitutea part of this specification and include exemplary embodiments of thepresent invention and illustrate various objects and features thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete understanding of the present invention may be obtainedby references to the accompanying drawings when considered inconjunction with the subsequent detailed description. The embodimentsillustrated in the drawings are intended only to exemplify the inventionand should not be construed as limiting the invention to the illustratedembodiments.

FIG. 1 is a graph showing mean epinephrine influx (μg/cm²/hr) obtainedfrom the tested formulations; epinephrine nanoparticles suspension(Epi-NP Susp) (size 200 nm), epinephrine solution (Epi-HBCD Sol),epinephrine suspension (Epi-CMC Susp), and epinephrine bitartratesolution (Epi Bit Sol).

FIG. 2A is a Fourier Transform Infrared (FT-IR) spectrum for epinephrinebase nanoparticles after fabrication (processing).

FIG. 2B is a FT-IR spectrum for epinephrine base nanoparticles beforeprocessing.

FIG. 3 illustrates particle size distribution of epinephrine basemeasured before size reduction (processing) using Mastersizer.

FIG. 4 illustrates particle size distribution of epinephrine basemeasured after size reduction using NiComp 370.

FIG. 5 is a FT-IR spectrum for epinephrine bitartrate nanoparticlesbefore and after processing (nanoparticle fabrication).

FIG. 6A is a graph showing the AUC (mean cumulative epinephrineconcentration) (μg/ml) obtained from the four tested formulations;Epi-NP Susp, Epi-CMC Susp, Epi-HBCD Sol, and Epi Bit Sol.

FIG. 6B is a graph showing mean epinephrine influx (μg/cm²/hr) obtainedfrom the tested formulations; Epi-NP Susp, Epi-CMC Susp, Epi-HBCD Sol,and Epi Bit Sol.

FIG. 7 is a graph showing Disintegration Time (DT) per second of tendifferent epinephrine 40 mg sublingual tablet formulations.

FIG. 8 is a graph showing Percent of Drug Released (DR %) of tendifferent epinephrine 40 mg sublingual tablet formulations.

FIG. 9A is a graph showing Percent of Drug Released (DR %) forepinephrine 40 mg sublingual tablet formulations 10, 9, and 2.

FIG. 9B is a graph showing Area under the Curve (AUC) for epinephrine 40mg sublingual tablet formulations 10, 9, and 2.

FIG. 10 is a graph showing the effect of chitosan to tripolyphosphate(TPP) weight ratio and epinephrine load on the nanoparticle size.

FIG. 11 is a graph showing the effect of chitosan to TPP weight ratioand epinephrine load on the nanoparticle zeta potential.

FIG. 12 is a graph showing the effect of chitosan to TPP weight ratioand epinephrine load on epinephrine encapsulation efficiency.

FIG. 13 is a graph showing the effect of chitosan to TPP weight ratioand epinephrine load on nanoparticle fabrication yield.

FIG. 14 is a graph showing the effect of the pH of the medium onepinephrine nanoparticle characteristics using 40% Epi theoretical load.

DETAILED DESCRIPTION OF THE INVENTION

For the purpose of promoting an understanding of the principles of theinvention, reference will now be made to embodiments illustrated hereinand specific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of theinvention is thereby intended. Any alterations and further modificationin the described compositions and methods and any further application ofthe principles of the invention as described herein, are contemplated aswould normally occur to one skilled in the art to which the inventionrelates.

Example 1 One Embodiment of Epinephrine Sublingual Tablet FormulationSummary of In Vitro Diffusion Experiments and Results

The experiments described herein were carried out to assess the in vitrodiffusion of epinephrine nanoparticles. The use of epinephrinenanoparticles instead of epinephrine salt was hypothesized to enhancethe sublingual bioavailability of epinephrine from administration of afast-disintegrating sublingual tablet formulation for the emergencytreatment of anaphylaxis and/or treatment of other conditions responsiveto epinephrine.

Methods:

The diffusion of 8 μg epinephrine from four formulations, epinephrinebase nanoparticles suspension (Epi-NP Susp) (size 200 nm), epinephrinesolution (Epi-HBCD Sol); epinephrine base usinghydroxypropyl-β-cyclodetrin as a solubilizing agent, epinephrinesuspension (Epi-CMC Susp); epinephrine base using 0.3% carboxymethylcellulose as a suspending agent, and epinephrine bitartrate solution(Epi Bit Sol), was studied over 8.5 hours using automated flow-throughFranz cell system (n=6). Cumulative epinephrine concentrations in thereceptor cells were measured using HPLC-UV (High Performance LiquidChromatography system with an ultraviolet detector). The cumulativeepinephrine concentration versus time (AUC), maximum epinephrine flux(J_(max)), time to reach Jmax (Jt_(max)), and epinephrine permeationcoefficient (Kp) for each formulation were calculated and statisticallyanalyzed using one-way ANOV and Tukey-Kramer tests, NCSS program, at alevel of significance p<0.05.

Results:

The AUC and Jmax obtained from epinephrine nanoparticles (Epi-NP Susp),10.4±1.7 μg/ml/hr and 15.1±1.9 μg/cm²/hr respectively, weresignificantly higher than epinephrine suspension (Epi-CMC Susp), 5.1±1.1μg/ml/hr and 10.4±1.6 μg/cm²/hr, epinephrine solution (Epi-HBCD Sol),5.5±0.5 μg/ml/hr and 8.6±0.3 μg/cm²/hr, and epinephrine bitartrate (EpiBit Sol), 4.6±0.9 μg/ml/hr and 7.9±1.0 μg/cm²/hr. Jt_(max) was notsignificantly different between the four formulations. The Kp ofepinephrine nanoparticles, 0.19±0.07 cm/hr was significantly higher thanepinephrine suspension, 0.13±0.002 cm/hr, epinephrine solution,0.11±0.04 cm/hr, and epinephrine bitartrate, 0.10±0.04 cm/hr. Theseresults are illustrated in the graph of FIG. 1.

Conclusions:

In these experiments, the permeation of epinephrine nanoparticles(Epi-NP Susp) was almost 2 folds higher than the epinephrine bitartrate(Epi Bit Sol) and epinephrine solution (Epi-HBCD Sol). Epinephrinenanoparticles may have the potential to enhance the sublingualbioavailability of epinephrine compared to epinephrine salt insublingual tablet formulation. Ex vivo and in vivo studies arecontemplated and will be pursued to confirm these results.

Details of Fabrication Experiments and Results Fabrication ofNanoparticles

Nanoparticles were fabricated from epinephrine base or epinephrinebitartrate (Bit) using high energy fluidization (microfluidization)techniques. These techniques involve the use of an epinephrinesuspension of various solvents, particularly water or isopropanol, atvarious pressures ranging from about 8,000 psi to 30,000 psi for variouspasses. Particles sizes were measured before and after size reductionusing a Mastersizer (Malvern) and/or a NiComp 370 Submicron ParticleSizer (NiComp). The particles were lyophilized (freeze-dried) using abench top lyophilizer (ART Inc.).

Solubility Studies

In order to determine suitable vehicles to suspend epinephrine base andepinephrine bitartrate (Bit) for nanoparticle fabrication, solubilitystudies were carried out to select the vehicles that minimallysolubilize the drug.

TABLE 1 Solubility Amount Sample Name Dissolved (μg/mL) Epinephrine Basesolubility in water 53.37 Epinephrine Bit solubility in methanol 209.01Epinephrine Bit solubility in isopropyl alcohol 12.30 Epinephrine Bitsolubility in acetonitrile 15.46 Epinephrine Bit solubility in acetone31.28 Epinephrine Bit solubility in hexane 0.53 Epinephrine Bitsolubility in choloroform 1.87 Epinephrine Bit solubility intetrahydrofuran (THF) 155.18 Epinephrine Bit solubility in ethyl acetate12.60Fabrication of nanoparticles was first attempted using epinephrine base

Fabrication: Epinephrine Base

TABLE 2 Epinephrine Base Particle Size Sample Pressure DistributionStandard Sample Color Concentration (psi) (nm) Deviation Temperatureafter Sample Solvent (mg/ml) (#passes) (NiComp) (SD) (° C.) Processing 1water 0.3 30,000 273.9 179 43.3 brown 2 water 0.308 29,000 334 276 18.3brown (1) 3 0.1% 1.03 15,000 335 41 36.8 brown phosphoric (2) first passacid 41.1 second pass 4 1 M acetic 1.55 8,500 392 247 36.6 brown Acid(1) first pass 15,000 38.4 (1) second pass 5 water 4.03 15,000 905.9 8210 brown 6 0.1 mM 4.02 15,000 903.1 97 8.3 brown sodium metabisulfite inwater 7 0.1 mM 12.02 15,000 903.1 326 8.3 pink sodium Note: 111.5metabisulfite nm (80)%) in 0.1 M and 2.2 nm perchloric (20%) acid usingZetasizer achine

Nanoparticles of epinephrine base in various sizes were produced rangingin diameter from about 273.9 to 905.9 nm.

First Sample

The sample consisted of 30 mg epinephrine in 100 ml of distilled water.One pass at 30,000 psi was applied and a temperature of 43.3° C. wasmeasured after the process. The sample was processed using a M-110P HighEnergy Fluidizer™ (Microfluidics). The particles were lyophilized usingbench top lyophilizer (ART Inc.). The mean particle size obtained was273.9 nm using the NiComp 370 Submicron Particle Size Analyzer. Thesample was stored in the refrigerator.

Second Sample

This sample consisted of 30 mg epinephrine in 100 ml of distilled water.One pass at 29,000 psi was applied and a temperature of 18.3° C. wasmeasured after the process. The homogenizer was setup using the coolingcoil. Ice packs and tap water were used to cool the pressurized sampleto 14° C. The mean particle size obtained was 334.3 nm using the NiComp370 Submicron Particle Size Analyzer. The sample was stored in therefrigerator.

Third Sample

This sample was prepared in 0.1% phosphoric acid. The phosphoric acidsolution was prepared by diluting 0.5 ml of phosphoric acid 85%(Mallinckrodt Chemicals, LOT H39A04, Exp. Sep. 30, 2011) in 500 ml ofdistilled water. The epinephrine sample was prepared by weighing 103 mgof epinephrine base into 100 ml of 0.1% phosphoric acid solution priorto sample passes. Two passes at 15,000 psi were applied to the sample.In the first pass a temperature of 36.8° C. was measured after theprocess and in the second pass a temperature of 41.1° C. was obtained.The mean particle size obtained was 334.6 nm using the NiComp 370Submicron Particle Size Analyzer. The sample was stored in therefrigerator.

Fourth Sample

This sample was prepared in 1M acetic acid. The 1M acetic acid solutionwas prepared by diluting 27.5 ml of glacial acetic acid (BDH Aristar,ACS, USP, FCC grade, LOT 200929924) in 500 ml of distilled water. Theepinephrine sample was prepared by weighing 155 mg of epinephrine baseinto 100 ml of 1M acetic acid solution. The M-110p was flushed withdistilled water, followed by acetic acid solution prior to samplepasses. Two passes were applied to the sample, in the first pass apressure of 8,500 psi was applied and a temperature of 36.6° C. wasmeasured in the collected sample. In the second pass a pressure of15,000 psi was applied and a temperature of 38.4° C. was measured in thecollected sample. The mean particle size obtained was 392.0 nm using theNiComp 370 Submicron Particle Size Analyzer. The sample was stored inthe refrigerator.

Fifth, Sixth, and Seventh Samples

These samples were prepared in a dark room to avoid light. Thehomogenizer was setup using the cooling coil. Ice packs and tap waterwere used to cool the pressurized samples. Higher drug concentration wasused in the seventh sample since the acidic solvent tends to dissolvemore drug than the other previously-used solvents.

Visual Observations

The main problem was discoloration (a brown color formed) due todegradation. All samples were discolored to a pinkish color and thenbecame dark brownish after processing, indicating epinephrineinstability. The seventh sample (water+0.1 mM sodium metabisulfite+0.1 Mperchloric acid) discolored to a slightly pinkish color. 0.1 mM sodiummetabisulfite+0.1 M perchloric acid usually provided optimum stabilityfor epinephrine for several months.

The FT-IR spectrum for epinephrine base before (FIG. 2B) is differentfrom the FT-IR spectrum after processing (FIG. 2A), which reflects thedegradation that occurs during processing. The epinephrine base requiredstabilization with acetic acid or phosphoric acid (in the suspensionmedia) and cooling of the reaction chamber to minimize degradation.

Sizing

The first sample (epinephrine in water) was used.

TABLE 3 Sizes of Epinephrine Base Before and After Processing AfterFabrication Sample Before Fabrication (nm) (110 F., 30 Kpsi) (nm) 133030 273.9 2 32530 3 33160 Mean 32900 Standard Error 192.04 StandardDeviation 332.62 179

The epinephrine particle size reduction to nanosize was successful. Themean±SD size was reduced from 32.91±0.33 μm (FIG. 3) to 273.9±179.0 nm(FIG. 4).

Fabrication: Epinephrine Bitartrate (Bit)

In light of the instability associated with the epinephrine baseparticles, fabrication using the epinephrine salt, epinephrinebitartrate, was pursued.

Isopropyl alcohol (IPO) was selected as a suspending vehicle based onits safety profile and the solubility study previously performed (seeabove) for several solvents.

TABLE 4 Epinephrine Bitartrate (Bit) Pressure Standard Concentration(psi) PSD nm Deviation Sample Solvent (mg/ml) (# passes) (NiComp) (SD) 1IPO 7.0 15,000 (1) 43,000 >20,000 2 IPO 3.5 25,000 (1) 8,766 NA 25,000(1) 3,879 3 IPO 0.875 25,000 (1) 3,971 2032 4 IPO 0.70 25,000 (6) 2,3682065  25,000 (16) 1,203 924The mean±SD size was reduced from 150,700±5000 nm to 1,203±924 nm.

Observations

Nanoparticles of epinephrine bitartrate in various sizes were producedranging in diameter from about 43,000 to 1,203 nm.

The first sample, a suspension of 7.0 mg/ml, was used as a stocksuspension and was used to prepare the other dilutions. Thus, the passesare additive and each (pass) represents an additional pass to theprevious dilution.

After ten passes in the last run, includes samples one, two, three, andthe first pass of sample 4, the particle size distribution (PSD) did notchange (no effect after ten passes) according to NiComp readings.

The fourth sample was processed six times (6 passes in one step)followed by an additional ten passes (for a total of sixteen passescontinuously).

The epinephrine bitartrate (salt form of epinephrine) was more stablethan the epinephrine base, did not show any discoloration, and toleratedthe fabrication conditions (nanomilling).

First Sample

The particle size distribution (PSD) of epinephrine bitartrate afterprocessing (fabrication) using Zetasizer was 5000 nm (60%) and 500-1000nm (30-40%). The yield of fabricated epinephrine bitartrate after dryingwas 68%. The Fourier Transformation Infrared (FT-IR) spectrums aresimilar in both epinephrine bitartrate before and after processing (FIG.5).

Details of In Vitro Diffusion Experiments and Results

Epinephrine diffusion was evaluated using an automated, flow throughcell system (n=6) under the following parameters:

Flow rate: 50 μl/minute

Donor cell orifice area: 0.2 cm²

Sample volume added to donor cell: 200 μl

Medium in receptor cells: phosphate buffer (pH=5.8)

Membrane: 7 Spectra/Por® dialysis membranes (1000 MWt cutoff).

Epinephrine, base or salt equivalent to 400 μg/ml epinephrine base, inthe following four different formulations were used:

-   -   1) Epinephrine base nanoparticles suspension (Epi-NP Susp).    -   2) Epinephrine base suspension using 0.3% carboxymethyl        cellulose as a suspending agent (Epi-CMC Susp).    -   3) Epinephrine base solution using hydroxypropyl-β-cyclodetrin        as a solubilizing agent (Epi-HBD Sol).    -   4) Epinephrine bitartrate solution (Epi Bit Sol).

200 μl from each of the four formulations was spiked into the donorcells. Samples were collected every 30 minutes for 8.5 hours andanalyzed by High Performance Liquid Chromatography (HPLC) forepinephrine concentration.

HPLC Analysis

HPLC analysis was performed under the following parameters:

PerkinElmer HPLC system with ultraviolet (UV) detector

Column: Econspher (Alltech), C₁₈ 4.6×150 mm, 3 μm

Mobile Phase: USP 26^(th) Edition, 2003

Flow Rate: 1 ml/minute

Detection Wavelength: 280 nm

Retention Time: epinephrine 4.8 minutes

Statistical Analysis of Results

Results were statistically analyzed using one-way ANOV and Tukey-Kramertests, NCSS program, at a level of significance p<0.05.

Mean±SD values of cumulative epinephrine concentration versus time(AUC), maximum epinephrine flux (JMax), time to reach JMax (tJMax), andepinephrine permeation coefficient (Kp) for each formulation wascalculated.

Results

Mean±SD values of cumulative epinephrine concentration versus time(AUC), maximum epinephrine flux (JMax), and epinephrine permeationcoefficient (Kp) obtained from Epi-NP Susp were significantly higherthan Epi-CMC Susp, Epi-HBCD Sol, and Epi Bit Sol (p<0.05). The time toreach JMax (tJMax) was not significantly different between the fourformulations. These results are illustrated in the graphs of FIGS. 6A-B.

TABLE 5 In Vitro Diffusion Data Formulation Epi-NP Susp Epi-CMC SuspEpi-HBCD Sol Epi Bit Sol AUC (μg/ml/hr) 10.4 ± 1.7* 5.1 ± 1.1 5.5 ± 0.54.6 ± 0.9 JMax (μg/cm²/hr) 15.1 ± 1.9* 10.4 ± 1.6  8.6 ± 0.3 7.9 ± 1.0t_(Jmax) (hr) 9.41 ± 0.26 9.41 ± 0.50 10.17 ± 0.10  10.12 ± 0.09  Kp(cm/hr)  0.19 ± 0.07*  0.13 ± 0.002 0.11 ± 0.04 0.10 ± 0.04

Example 2 Second-Generation Epinephrine Sublingual Tablet FormulationsSummary Purpose

In vivo bioavailability of epinephrine (Epi) following sublingual (SL)administration of a 40-mg dose from different first-generation (Gen1)fast-disintegrating tablet formulations is affected by the tabletexcipients (Rawas-Qalaji et al. Biopharm Drug Disposition 27 (9):427-4352006). The second-generation (Gen2) SL tablets of Epi were designed toevaluate the effect of grade and proportion of excipients on the invitro characteristics of the tablets, especially the percentage of Epireleased from the tablet (dissolution), which was found to be affectedby the tablet excipients in the first-generation tablets (Gen1).

Methods

Epi 40-mg SL tablet formulations (F), Gen1 (F2, F9, and F10), and Gen2(F1 and F3-8) containing 0-25% mannitol (M), using a single grade orcombinations of grades of microcrystalline cellulose (MCC: KG-802,PH-301, PH-M-06), were prepared by direct compression. The effect ofadding citric acid for taste-masking was also evaluated. Allformulations were evaluated for USP weight variation (WV) and contentuniformity (CU). Disintegration times (DT) and dissolution at 60seconds, expressed as percent of drug released (% DR), were determinedusing procedures that simulated the SL administration site (AAPSPharmSciTech 12:544-552 2011).

Results

All ten formulations were within USP limits for WV and CU. Dissolutiontimes (DT) of all formulations were <20 seconds. Incorporation of up to15% mannitol (M) into tablet formulations (Gen2, F1, F3, F4, F5, F7) didnot affect % DR, but it decreased significantly (p<0.05) when M load wasincreased to 25% (Gen1, F9, F10; and Gen2 F6) and 25% (Gen2,e). At M upto 15%, the incorporation of the MCC grades PH-301 (Gen1, F2; and Gen2,F1, F3, F4, F5, F7) and/or PH-M-06 (Gen1, F9; and Gen2, F1, F4, F5, F7)resulted in higher % DR compared to KG-802 (Gen2, F8). Gen2, F7 thatcontained PH-301 and PH-M-06 at ratio 6:1 and citric acid for tastemasking resulted in virtually complete % DR.

Conclusion

Second generation formulations that contain mannitol at 15% as asweetening agent and to enhance the mouthfeel of the tablet, acombination of two MCC grades (PH-301: PH-M-06) at 6:1, and a citricacid to mask the taste of Epi resulted in SL Epi tablet formulationswith optimal DT and % DR.

Introduction

In a validated animal model, first-generation (Gen1) 40 mgfast-disintegrating Epi tablet formulation and a 0.3 mg Epi fromintramuscular (TM) injection resulted in similar Epi plasmaconcentrations (J Allergy Clin Immunol 117:398-403 2006).

For selection of the optimal first-generation (Gen1) formulations for invivo evaluation in the validated animal model, disintegration time (DT)was used as the primary in vitro procedure. However in previous in vivoand in vitro studies, the sublingual administration of a 40 mg dose fromdifferent first-generation (Gen1) fast-disintegrating tabletformulations with similar DTs resulted in different bioavailabilities(Rawas-Qalaji et al. Biopharm Drug Disposition 27 (9):427-435 2006). Itwas noted that the excipients affect the rate and extent of epinephrinedissolution and therefore its bioavailability. Dissolution assessment isa more selective in vitro test that should be used as a potentialpredictive tool for the in vivo results. Thus, second-generation (Gen2)epinephrine sublingual tablet formulations were developed by evaluatingthe effect of the grade and proportion of excipients in testformulations on epinephrine dissolution.

The instant inventors developed a unique dissolution apparatus thatsimulates the conditions in the sublingual cavity (Rachid, O. et al.AAPS PharmSciTech 12(2):544-552 2011) and overcomes the problemsassociated with the use of an official USP dissolution apparatus(USP/NF. Physical Tests: Dissolution (711); 22/17 ed. Rockville, Md.:United States Pharmaceutical Convention Inc; 2007) for sublingualtablets. The limited volume of saliva produced over a short period oftime in a relatively static environment is the condition in thesublingual cavity that was simulated in the unique dissolutionapparatus. It enabled the instant inventors to discriminate amongepinephrine sublingual formulations with similar DTs. Using the uniquedissolution apparatus, the rate and extent of release of epinephrine wasmeasured to determine the quantity and quality of non-medicinalingredients (NMIs) that can be added without inhibiting the release anddissolution of epinephrine. Based on these dissolution results, the bestperforming epinephrine sublingual formulations will be selected for thein vivo studies in the validated animal model to generate in vivo data.

It has been shown using in vitro methods that the quantity and grade ofsoluble or in-soluble NMIs that can be included into the epinephrinesublingual tablet can exert major effects on the tablet characteristicsand the rate of drug release (dissolution). These changes affect therate and extent of epinephrine sublingual absorption. These NMIs includea wide range of grades of insoluble diluents, microcrystalline cellulose(MCC), disintegrates, soluble sweetening and flavoring agents to maskthe bitter taste of epinephrine, and secretagogues (agents that promotesaliva secretion). The secretagogues can enhance tablet disintegrationby promoting saliva excretion which can improve tablet dissolution andepinephrine release, and should promote epinephrine absorption.

Accordingly, the second-generation epinephrine sublingual tablets wereformulated with the main objective of the development of the bestperforming tablet using in vitro studies. The in vitro assessment of thesecond-generation epinephrine sublingual tablets included the evaluationof weight variation (WV), content uniformity (CU), hardness (H),disintegration time (DT), wetting time (WT), dissolution (percent ofdrug released, % DR), and taste improvement.

The extent of the bitter taste of epinephrine is unknown raising concernabout patient acceptability of a sublingual tablet of epinephrine,especially by children. An electronic tongue (e-tongue) was utilized toassess and predict the bitterness intensity of epinephrine on abitterness scale. Furthermore, the effect of NMIs, such as sweeteners(e.g. aspartame and acesulfame potassium) and flavors (e.g. citric acid)on the overall taste of the epinephrine sublingual tablets was evaluatedand the masking benefit of these NMIs on the bitter taste of epinephrinewas measured (Rachid, O. et al. AAPS PharmSciTech 11(2):550-557 2010).

Based on the results from the e-tongue and using the unique dissolutionassembly, the best performing second-generation epinephrine sublingualtablet formulations were selected for the new series of in vivobioavailability studies using the validated animal model (Rawas-Qalajiet al. Biopharm Drug Disposition 27 (9):427-435 2006). All of thechanges in the second-generation epinephrine sublingual formulationsthat demonstrated improved epinephrine dissolution from in vitro resultswill be confirmed in vivo using the validated rabbit model to study theepinephrine bioavailability after sublingual absorption.

Hypothesis for Second-Generation Formulation Study:

the second-generation epinephrine sublingual tablets that showed >98%release of medication within 60 seconds (dissolution) will demonstrateboth increased rate and extent of epinephrine absorption in thevalidated animal model after sublingual administration as compared tothe first-generation formulations.

Methods:

The optimal first-generation 40 mg epinephrine formulation, tested inthe validated animal model (Rawas-Qalaji et al. Biopharm DrugDisposition 27 (9):427-435 2006) was used as the model sublingualformulation since it resulted in epinephrine serum concentrations notsignificantly (p<0.05) different from those obtained from a 0.3 mgepinephrine autoinjector.

The effects of varying concentrations of mannitol, and various gradesand percentages of microcrystalline cellulose diluents on dissolution(percent of epinephrine dose released and dissolved in 60 seconds) werestudied using the novel dissolution apparatus (Rachid, O. et al. AAPSPharmSciTech 12(2):544-552 2011).

The effect of the addition of citric acid on masking the bitter taste ofepinephrine was evaluated using the electronic tongue (Rachid, O. et al.AAPS PharmSciTech 11(2):550-557 2010).

The effect of increasing the weight and dimensions of the sublingualepinephrine tablet formulation to increase the surface area and improvedissolution was also evaluated using the novel dissolution apparatus(Rachid, O. et al. AAPS PharmSciTech 12(2):544-552 2011).

The in vitro re-assessment of the first-generation and assessment of thesecond-generation epinephrine sublingual tablets included the evaluationof weight variation (WV), content uniformity (CU), hardness (H),disintegration (DT), wetting times (WT), dissolution (percent of drugreleased, % DR), using the novel dissolution apparatus (Rachid, O. etal. AAPS PharmSciTech 12(2):544-552 2011), and taste-masking (Rachid, O.et al. AAPS PharmSciTech 11(2):550-557 2010) during the development ofthese tablets.

Results and Discussion:

Of the numerous first and second generation 40 mg epinephrine sublingualtablets formulated and tested, the results of the ten best prospectswere selected based on in vitro test results as reported in Tables 6A-Dand Table 7 and FIGS. 7 and 8.

TABLE 6A Formulation Contents: Forumulation 1 2 3 4 5 6 7 8 9 10Epinephrine 72.77 72.77 72.77 72.77 72.77 72.77 72.77 72.77 72.77 72.77bitartrate (mg) MCC PH-301 74.48 66.81 46.56 40.83 33.06 66.80 (mg) MCCPH-M-06 11.17 46.56 40.83 11.17 33.06 (mg) MCC KG-802 53.31 (mg)Pharmaburst 74.23 (mg) Mannitol 22.50 22.50 30.00 37.50 15.00 37.50Pearlitol (15%) (15%) (15%) (25%) (10%) (25%) 400 DC (mg)/(%) Mannitol25.57 34.10 Ludiflash (11%) (15%) (mg)/(%) Citric Acid 2.50 2.50 2.50(mg) L-HPC LH-11 9.51 7.42 5.17 5.17 9.07 3.67 8.66 5.92 3.67 (mg)Magnesium 4.00 3.00 3.00 3.00 4.00 3.00 4.00 3.00 3.00 3.00 stearate(mg) Total Tablet 200.00 150.00 150.00 150.00 200.00 150.00 200.00150.00 150.00 150.00 Weight (mg)

TABLE 6B Tablet Dimensions 1 2 3 4 5 6 7 8 9 10 Diameter (mm) 10.00 8.508.50 8.50 10.00 8.50 10.00 8.50 8.50 8.50 Thickness (mm)  2.00 1.75 1.751.75  2.00 1.75  2.00 1.75 1.75 1.75

TABLE 6C In Vitro Characteristics 1 2 3 4 5 Compression 21.00 18.5018.50 18.25 19.50 Force (kN) Hardness (kg)- 2.96 ± 0.04  1.1 ± 0.04 1.23± .23  1.45 ± 0.03 2.67 ± 0.08 mean ± SEM Weight Variation (mg)- 201.9 ±0.64  147.22 ± 0.26  148.36 ± 0.31  148.39 ± 0.2   199.4 ± 0.69  mean ±SEM Content Uniformity 99.04 ± 0.67  103.79 ± 0.59  99.64 ± 0.78  100.08± 0.44  98.55 ± 0.56  (%)- mean ± SEM Disintegration 9.17 ± 0.31 10.67 ±0.42  10.5 ± 0.22 12.67 ± 0.21    13 ± 0.55 Time (sec)- mean ± SEMPercent of Drug Released at 60 80.63 ± 2.42  98.66 ± 2.38  79.77 ± 1.58 96.41 ± 0.75  88.39 ± 2.15  Sec (%)- mean ± SEM In Vitro Characteristics6 7 8 9 10 Compression 18.25 22-22.5 20.50 17.75 19.25 Force (kN)Hardness (kg)- 1.23 ± 0.01 3.03 ± 0.07 1.22 ± 0.02 1.23 ± 0.01 1.23 ±0.01 mean ± SEM Weight Variation (mg)- 152.32 ± 0.33  202.0 ± 0.81 148.64 ± 0.23  149.55 ± 0.35  149.69 ± 0.46   mean ± SEM ContentUniformity 100.03 ± 0.48   97.82 ± 1.02  99.89 ± 0.23  98.04 ± 0.4 99.88 ± 0.21  (%)- mean ± SEM Disintegration 13.17 ± 0.31  13.5 ± 0.7614.83 ± 0.4  15.33 ± 0.33  18.5 ± 0.34 Time (sec)- mean ± SEM Percent ofDrug Released at 60 68.65 ± 1.08  99.8 ± 0.41 53.46 ± 2.29  77.59 ±2.16  67.54 ± 1.59  Sec (%)- mean ± SEM SEM: Standard Error of Mean

TABLE 6D In Vivo Characteristics 1 2 3 4 5 6 7 8 9 10 Area under theCurve, AUC 0-3 h 1861 ± 537  615 ± 87  646 ± 202 (ng/mL/min)-mean ± SEMCmax (ng/mL)- 31 ± 13   6 ± 0.9 6.7 ± 3.2 mean ± SEM Tmax (min)- 9 ± 228 ± 10 16 ± 4  mean ± SEM

Table 7 shows disintegration time (DT) versus Percent of Drug Released(DR %) at 60 seconds of ten different epinephrine 40 mg sublingualtablet formulations.

TABLE 7 Formulations DT (9-19 sec) % DR at 60 sec (53-100%) 1 9 81 2 1199 3 11 80 4 13 96 5 13 88 6 13 69 7 14 100 8 15 53 9 15 78 10 19 68

All tablets met the criteria for hardness (H), weight variation (WV) andcontent uniformity (CU) and exhibited disintegration times (DT) between9 and 19 seconds (Table 6C; Table 7; FIG. 7; and FIG. 8).

These results indicate that the in vitro method to test drug releasefrom sublingual tablets using the unique dissolution apparatus was ableto discriminate among formulations which showed similar in vitro data,i.e. disintegration time.

It can be seen from the preliminary data from animal studies that forfirst-generation formulations 2, 9 and 10, there is some correlationbetween percent dissolved (% DR), in vitro data, and Area Under thePlasma Concentration versus Time Curve (AUC), in vivo data. This data isreported in Table 8 and FIGS. 9A-B.

TABLE 8 In vitro in vivo Rank Correlations In vitro In vitro DRFormulation DT (sec) % at 60 sec SEM In vivo AUC* SEM 10 18.5 67.54 1.59646 202 9 15.33 77.59 2.16 615 87 2 10.67 98.66 2.38 1861 537 *Comparedto in vivo AUC of Epipen 0.3 mg (2431 ± 386)

Although first-generation formulations 2, 9, and 10 (see Summary) sharesimilar in vitro disintegration times (DT) of less than 20 seconds, theyresulted in widely different bioavailability. In general, disintegrationtime (DT) is considered to be a poor indicator of in vivobioavailability. A more predictive in vitro method was required toreflect in vivo behavior. The in vitro drug release method carried outusing the unique dissolution apparatus was able to differentiate betweenthese formulations in a rank correlation. The ones that failed in vivoresulted in a DR % of less than 80%. However, the best performing invivo formulation, formulation 2, resulted in the best DR % of almost100%.

From the in vitro data (Table 7; FIG. 7; FIG. 8), it can be seen that asecond-generation formulation 7 is the optimal formulation. Thecomposition and characteristics of second-generation formulation 7 areshown in Tables 9A-E. The in vivo evaluation of the bioavailability ofsecond-generation formulation 7, using the validated animal model, willbe the next study undertaken.

TABLE 9A The Second Generation of Epinephrine Sublingual Tablets(Formulation 7) Tablet Weight Percentage Contents Ingredients Type (mg)% 1 Active *epinephrine bitartrate 72.77 36.39 Ingredient 2 Filler**Ceolus, MCC 11.17 5.59 (PH-M-06) 3 Filler **Ceolus, MCC 66.80 33.40(PH-301) 4 Filler ***Ludiflash 34.10 17.05 (88% mannitol) 5 Flavor†Citric Acid 2.50 1.25 6 Disintegrant ‡L-HPC (LH-11) 8.66 4.33 7Lubricant Mg Stearate 4.00 2.00 Tablet Weight 200.00 100.00 *Each tabletcontained 72.77 mg Epinephrine bitartrate which is equivalent to 40 mgEpinephrine base. **Ratio of MCC (PH-301) and MCC (PH-M-06) was kept at6:1. ***Ludiflash consists of average 88% mannitol, Ludiflash at 17.05%will contain 15% mannitol. †Ratio of Epinephrine bitartrate and citricacid was kept as 29:1. ‡Ratio of total MCC and L-HPC was kept as 9:1.MCC (PH-M-06) particle size is 7 μm. MCC (PH-301) particle size is 50μm. Ludiflash particle size is 200 μm.

TABLE 9B Tablet Press Die Size: 13/32″ Die Shape: Round Punch Surface:Flat CF (kN): 22

TABLE 9C Tablet Dimensions Diameter: 10 mm Height:  2 mm

TABLE 9D Tablet H Characteristics WV (mg) CU (%) (kg) DT (sec) DR % (60sec) Mean 202.00 97.82 3.03 13.50 99.80 SD 2.58 3.23 0.17 1.87 1.17 RSD(CV) 1.28 3.30 5.61 13.86 1.18 SEM 0.97 1.02 0.07 0.76 0.41

TABLE 9E Key for Abbreviations Used throughout the Specification MCC:microcrystalline cellulose DT: disintegration time L-HPC:low-substituted DR %: percent of drug released hydroxypropyl celluloseWV: weight variation SD: standard deviation CU: content uniformity RSD:relative standard deviation H: hardness CV: coefficient of variationSEM: standard error of mean

Improvements in Tablet Formulations (Second Generation Compared to FirstGeneration)

1. larger tablet surface area to boost API dissolution and absorption2. harder tablets to withstand shipping and handling3. taste enhanced tablets to mask bitter taste of epinephrine4. improved tablet texture and mouthfeel to enhance patient compliance5. complete dissolution at 60 seconds

Example 3 Epinephrine Sublingual Tablet Formulations Using ChitosanSummary Objective:

to prepare epinephrine nanoparticles of optimum size and encapsulationefficiency using chitosan and tripolyphosphate (TPP).

Purpose:

Epinephrine was previously formulated into a fast-disintegratingsublingual tablet (AAPS PharmSciTech. 2006; 7(2): Article 41) and thesublingual bioavailability was established in a validated animal model(Rawas-Qalaji et al. J Allergy Clin Immunol 117:398-403 2006) for thepotential first-aid treatment of anaphylaxis in community settings. Thepurpose of this study is to develop and characterize epinephrinenanoparticles using chitosan as a polymer to enhance the sublingualbioavailability of epinephrine.

Methods:

Epinephrine bitartrate equivalent to epinephrine 10%, 20%, 30% and 40%were loaded into chitosan nanoparticles from crab shells using ionicgelation method. Chitosan to tripolyphosphate (TPP) weight ratio wasstudied at 2:1, 3:1, 4:1, 5:1 and 6:1. The medium's pH effect and thereproducibility of the fabrication process were evaluated. Particle sizeand zeta potential were measured immediately after preparation ofnanoparticles using zetasizer (Malvern). All samples were centrifuged at15000 rpm and the supernatant was analyzed using HPLC-UV to determinethe encapsulation efficiency of different weight ratios and epinephrineload. Fabrication yield was calculated from the collected and driednanoparticles. The mean size, mean zeta potential, encapsulationefficiency, and fabrication yield were plotted against weight ratio ofchitosan to TPP for each epinephrine load % and against the evaluated pHlevels.

Results:

Nanoparticles in the size range of 50-400 nm were obtained using 2:1 and3:1 weight ratios of chitosan to TPP. Zeta potential was increased withthe increase in weight ratio of chitosan to TPP, and decreased with theincrease in epinephrine load %. Encapsulation efficiency was increasedby increasing weight ratio of chitosan to TPP; but resulted in lowerencapsulation efficiency at 40% theoretical epinephrine load.Epinephrine nanoparticles fabricating at pH 2.75-2.85 resulted in thelowest particles size range. Mean±SD (RSD %) of particle size, zetapotential, epinephrine load, encapsulation efficiency, and fabricationyield for nanoparticles fabricated at 40% theoretical epinephrine load,2:1 chitosan to TPP weight ratio, and pH of 2.85 were 113±19 nm (17%),23±2 mV (10%), 28±2% (6%), 69.4% (6%), and 47±4% (9%), respectively.

Conclusion:

By adjusting the chitosan to TPP weight ratio and pH of the medium,optimum and reproducible size of epinephrine nanoparticles can beproduced. Encapsulation efficiency of epinephrine into chitosannanoparticles depends on weight ratio of chitosan to TPP and epinephrineload %.

Introduction

Epinephrine intramuscular (IM) injection in the thigh is the recommendedroute of administration for the first aid treatment of anaphylaxis inthe community. Due to several drawbacks of the injection alternativemethods of administration are being explored.

Examples 2 and 3 disclose fast-disintegrating tablets suitable forsublingual administration. Sublingual administration of 40 mgepinephrine as the bitartrate salt using these tablets resulted in arate and extent of epinephrine absorption similar to that achievedfollowing intramuscular injection of 0.3 mg epinephrine in the thigh.Sublingual doses ranging from 5 to 40 mg epinephrine as the bitartratesalt were evaluated to achieve equivalent plasma concentrations.

By fabricating epinephrine into nanoparticles and incorporatingpenetration enhancers and mucoadhesives (if needed) into the tabletformulation, the absorption of sublingually administered epinephrinewill significantly increase and will result in the reduction of thesublingual epinephrine dose required.

Chitosan Nanoparticle Fabrication

Epinephrine nanoparticles, 10%, 20%, 30%, and 40%, were fabricated by anionic gelation method using chitosan and tripolyphosphate (TPP) atweight ratios of 2:1, 3:1, 4:1, 5:1, and 6:1. The effect of the pH ofthe medium and the reproducibility of the fabrication were alsoevaluated.

An equivalent amount of epinephrine bitartrate, according to therequired theoretical load %, was dissolved in 4 mL deionized watersolution containing 3 mg tripolyphosphate (TPP).

Specific amounts of chitosan from crab shells (>75% deacetylated lowmolecular weight), according to the required ratio, was dissolved in 10mL acidified deionized water, pH 3 using acetic acid, by vortexing andbath sonication. Undissolved particles were removed by filtration.

The TPP solution containing epinephrine was added dropwise into thechitosan solution under continuous stirring using a magnetic stirrer andwas left to stir for approximately 30 minutes.

The formed particles were sized and the zeta potential measured.

Supernatant solution was collected, after centrifugation at 15,000 rpmand 15° C. for approximately 30 minutes, and analyzed for epinephrinecontent.

The formed pellets, after centrifugation, were washed with deionizedwater and centrifuged three times at 15,000 rpm and 15° C. forapproximately 30 minutes.

The pellets were then suspended with 1 mL deionized water and collectedfor lyophilization using a bench top lyophilizer (ART Inc.).

The particle size and zeta potential of the suspended nanoparticles weremeasured right after their fabrication and before centrifugation andfreeze drying using Zetasizer NanoZS90 (Malvern).

The same procedure was repeated at pH 2.85, 2.75, and 2.5 for 40%epinephrine theoretical load at 2:1 chitosan to TPP weight ratio toevaluate the effect of medium pH on nanoparticles characteristics.

The pH of 2.85 was then selected to repeat the fabrication proceduresthree times for 40% epinephrine theoretical load at 2:1 chitosan to TPPweight ratio to evaluate the reproducibility of the fabrication process.

Epinephrine amount encapsulated in the fabricated nanoparticles wascalculated indirectly from the epinephrine content in the collectedsupernatant solution.

Actual drug load (%), encapsulation efficiency (%), and yield (%) werecalculated according to the equations:

${{Actual}\mspace{14mu} {Drug}\mspace{14mu} {Load}\mspace{11mu} (\%)} = \frac{{amount}\mspace{14mu} {of}\mspace{14mu} {drug}\mspace{14mu} {in}\mspace{14mu} {nanoparticles} \times 100}{{amount}\mspace{14mu} {of}\mspace{14mu} {nanoparticles}}$${{Encapsulation}\mspace{14mu} {Efficiency}\mspace{11mu} (\%)} = \frac{{actual}\mspace{14mu} {drug}\mspace{14mu} {load} \times 100}{{theoretical}\mspace{14mu} {drug}\mspace{14mu} {load}}$${{Nanoparticles}\mspace{14mu} {Yield}\mspace{11mu} (\%)} = \frac{{nanoparticles}\mspace{14mu} {weight} \times 100}{{{drug}\mspace{14mu} {weight}} + {{polymer}\mspace{14mu} {weight}}}$

Epinephrine content in the collected supernatant solution was analyzedusing PerkinElmer HPLC system with UV detector and Econspher C₁₈,4.6×150 mm, 3 μm column (Alltech). Analysis and conditions wereperformed according to USP 26^(th) Edition, 2003 “Epinephrine InjectionMonograph.”

Results

Chitosan to tripolyphosphate (TPP) weight ratio used in the fabricationof the nanoparticles had a significant impact on the nanoparticle size(FIG. 10). Optimal sizes were obtained at 2:1 and 3:1 weight ratio ofchitosan to TPP for the various epinephrine loads (Table 10).

TABLE 10 Mean ± SD particles size of epinephrine nanoparticles weightratio of mean ± SD particle size (dnm) chitosan to TPP 10% Epi 20% Epi30% Epi 40% Epi 2:1  273 ± 110 155 ± 7  209 ± 2  289 ± 17 3:1 266 ± 92151 ± 4  139 ± 2  149 ± 17 4:1 444 ± 17 431 ± 64  356 ± 37 200 ± 4  5:1550 ± 88 685 ± 472 469 ± 59  487 ± 170 6:1  616 ± 352 1596 ± 1013  505 ±228 625 ± 20

The zeta potential of the nanoparticles increased with the increase ofchitosan to TPP weight ratio and decreased with the increase ofepinephrine load (Table 11; FIG. 11).

TABLE 11 Effect of Chitosan to TPP Weight Ratio and Epinephrine Load onthe Nanoparticle Zeta Potential weight ratio of Mean ± SD Zeta Potential(mV) chitosan to TPP 10% Epi 20% Epi 30% Epi 40% Epi 2:1 30 ± 02 22 ± 1120 ± 19 18 ± 08 3:1 44 ± 03 26 ± 05 34 ± 18 26 ± 34 4:1 49 ± 74 43 ± 0642 ± 29 31 ± 05 5:1 56 ± 16 46 ± 11 39 ± 24 39 ± 09 6:1 56 ± 05 55 ± 1433 ± 07 N/A

The encapsulation efficiency of epinephrine increased with the increaseof chitosan to TPP weight ratio and decreased with the increase of theepinephrine load (Table 12; FIG. 12).

TABLE 12 Effect of Chitosan to TPP Weight Ratio and Epinephrine Load onEpinephrine Encapsulation Efficiency weight Epinephrine EncapsulationEfficiency (%) ratio of chitosan to TPP 10% Epi 20% Epi 30% Epi 40% Epi2:1 91.6 79.8 67.5 61.0 3:1 91.5 87.2 71.5 65.7 4:1 90.5 95.8 77.2 69.45:1 94.7 99.9 82.5 68.9 6:1 108.1 105.0 112.5 69.7

The nanoparticle fabrication yield decreased with the increase of thechitosan to TPP weight ratio (Table 13; FIG. 13).

TABLE 13 Effect of Chitosan to TPP Weight Ratio and Epinephrine Load onNanoparticles Fabrication Yield weight Fabrication Yield (%) ratio ofchitosan to TPP 10% Epi 20% Epi 30% Epi 40% Epi 2:1 65.9 53.0 30.2 64.43:1 16.4 15.7 5.8 25.9 4:1 13.1 18.7 75.7 31.5 5:1 5.5 20.7 67 17.4 6:19.3 35.2 31.1 7.4

The size of the nanoparticles decreased dramatically with the decreaseof the pH of the chitosan solution from 3 to 2.85, 3.75, or 2.5 using40% epinephrine theoretical loaded (Table 14; FIG. 14).

TABLE 14 Effect of the pH of the Medium on Epinephrine NanoparticleCharacteristics Using 40% Epi Theoretical Load Encapsulation FabricationMean ± SD pH of chitosan Drug % Efficiency Yeild Particle Size solutionLoad (%) (%) (dnm) 3 28.7 71.5 117 689 ± 168  2.85 37.2 92.8 64.1 86 ±0.9 2.75 28.2 70.5 78.2 54 ± 0.4 2.5 25.5 63.7 77.8 96 ± 0.4

The nanoparticle fabrication at 40% epinephrine theoretical load and pH2.85 was reproducible (n=3). The mean±SD and RSD % of particle size,zeta potential, epinephrine load, encapsulation efficiency, andfabrication yield were 113±19 (17%), 23±2 mV (10%), 28±2 (6%), 69±4(6%), and 47±4 (9%), respectively.

Taken together the data show that optimum size of epinephrinenanoparticles and fabrication yield can be obtained by adjusting theweight ratio of chitosan to TPP. Further, nanoparticles zeta potentialand encapsulation efficiency of epinephrine depends on weight ratio ofchitosan to TPP and epinephrine load percent (%).

Ultimately, the carefully developed and critically evaluated sublingualepinephrine tablets described herein should have a major impact on themanagement of life-threatening anaphylaxis, especially in children.

All patents and publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.It is to be understood that while a certain form of the invention isillustrated, it is not intended to be limited to the specific form orarrangement herein described and shown. It will be apparent to thoseskilled in the art that various changes may be made without departingfrom the scope of the invention and the invention is not to beconsidered limited to what is shown and described in the specification.One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objectives and obtain theends and advantages mentioned, as well as those inherent therein. Thecompositions, epinephrine nanoparticles, pharmaceutical tablets,methods, procedures, and techniques described herein are presentlyrepresentative of the preferred embodiments, are intended to beexemplary and are not intended as limitations on the scope. Changestherein and other uses will occur to those skilled in the art which areencompassed within the spirit of the invention. Although the inventionhas been described in connection with specific, preferred embodiments,it should be understood that the invention as ultimately claimed shouldnot be unduly limited to such specific embodiments. Indeed variousmodifications of the described modes for carrying out the inventionwhich are obvious to those skilled in the art are intended to be withinthe scope of the invention.

1. A pharmaceutical composition comprising epinephrine nanoparticlesencapsulated with chitosan and tripolyphosphate (TPP), the compositionformulated for buccal or sublingual administration.
 2. Thepharmaceutical composition in accordance with claim 1, wherein theepinephrine is epinephrine bitartrate.
 3. (canceled)
 4. Thepharmaceutical composition in accordance with claim 1, furthercomprising at least one of a pharmaceutically-acceptable carrier, apenetration enhancer, and a mucoadhesive.
 5. The pharmaceuticalcomposition in accordance with claim 4, furthermore comprising at leastone of a taste enhancer and a sweetening agent and mouthfeel enhancer.6. The pharmaceutical composition in accordance with claim 5, whereinthe taste enhancer is citric acid and the sweetening agent and mouthfeelenhancer is mannitol. 7-15. (canceled)
 16. A method for fabricatingepinephrine nanoparticles comprising: a) preparing a solution includingchitosan, acetic acid, and water; b) preparing a solution includingtripolyphosphate (TPP) and epinephrine; and c) adding the solution ofstep b) to the solution of step a) and mixing together.
 17. The methodin accordance with claim 16, further comprising a step of adding atleast one of a taste enhancer and a sweetening agent and mouthfeelenhancer.
 18. The method in accordance with claim 17, wherein the tasteenhancer is citric acid and the sweetening agent and mouthfeel enhanceris mannitol.
 19. The method in accordance with claim 16, wherein theepinephrine is epinephrine bitartrate.
 20. An epinephrine nanoparticleproduced in accordance with the method of claim
 16. 21. A method forenhancing sublingual bioavailability of epinephrine in a subject in needthereof, comprising: providing a composition including epinephrinenanoparticles encapsulated with chitosan and tripolyphosphate (TPP) anda pharmaceutically-acceptable carrier; and administering the compositionto the subject.
 22. A method for treating a condition responsive toepinephrine in a subject in need thereof, comprising: providing acomposition including epinephrine nanoparticles encapsulated withchitosan and tripolyphosphate (TPP) and a pharmaceutically-acceptablecarrier; and administering the composition to the subject.
 23. Themethod in accordance with claim 22, wherein the condition is a cardiacevent or an allergic reaction.
 24. The method in accordance with claim23, wherein the cardiac event is cardiac arrest and the allergicreaction is anaphylaxis, asthma, or bronchial asthma.
 25. A method fortreatment of a breathing difficulty in a subject in need thereof,comprising: providing a composition including epinephrine nanoparticlesencapsulated with chitosan and tripolyphosphate (TTP) and apharmaceutically-acceptable carrier; and administering the compositionto the subject.
 26. The method in accordance with claim 25, wherein thebreathing difficulty is associated with anaphylaxis, asthma, bronchialasthma, bronchitis, emphysema, or respiratory infections.
 27. A methodfor treatment of an allergic emergency in a subject diagnosed with orsuspected of having an allergic emergency, comprising: providing acomposition including epinephrine nanoparticles encapsulated withchitosan and tripolyphosphate (TPP) and a pharmaceutically-acceptablecarrier; and administering the composition to the subject.
 28. Themethod in accordance with claim 27, wherein the allergic emergency isanaphylaxis, asthma, or bronchial asthma.
 29. A method for treatment ofa cardiac event in a subject diagnosed with or suspected of having acardiac event, comprising: providing a composition including epinephrinenanoparticles encapsulated with chitosan and tripolyphosphate (TPP) anda pharmaceutically-acceptable carrier; and administering the compositionto the subject.
 30. The method in accordance with claim 29, wherein thecardiac event is cardiac arrest. 31-41. (canceled)